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Molecular BiologyUniversity of Colorado

The main focus of my laboratory is to determine the role of Ras/MAPK signaling and Ets transcription factors in epithelial cell development and tumorigenesis, with a focus on pituitary and mammary model systems.

With regards to the pituitary project, we study how the combinatorial action of Ets factors, Ets-1 and GABP, acting in concert with the POU-homeodomain transcription factor, Pit-1, serve to regulate the lactotrope-specific basal and Ras-regulated expression of the prolactin gene. Utilizing biochemical, structural, molecular and transgenic approaches, we have discovered that Ets factors play a critical role in specifying lactotrope cell identity in pituitary ontogeny. Using transgenic mice, we show that targeting a dominant-negative Ets transgene to the pituitary lactotrope lineage abrogates pituitary lactotrope development. We are also studying the biological role of Pit-1ß, an alternative-splice form of Pit-1, which appears to act as a negative regulator of Pit-1 functions. We also seek to elucidate the molecular mechanisms by which lactotrope adenomas arise and why they rarely progress to frank carcinoma. To this end, we have generated GH4 pituitary cell lines stably expressing a doxycycline-regulated HA-V12Ras, to determine whether persistent vs short-term MAPK activation promotes cell proliferation, differentiation or apoptosis. Finally, we also plan to address the role of adult pituitary progenitor/stem cells in facultative lactotrope expansion during pregnancy and in tumorigenesis.

With regards to our breast cancer project, we study the role of the epithelial-specific Ets transcription factor, Ese-1, in human mammary epithelial cell tumorigenesis. Ese-1 is over-expressed in many breast cancers and, thus, it is particularly relevant in human disease. We have shown that Ese-1 confers a transformed phenotype to immortalized, but nontransformed MCF-12A human mammary epithelial cells via a novel cytoplasmic mechanism. We also demonstrated that exclusive cytoplasmic targeting of a unique, 40-AA serine- and aspartic rich (SAR) subdomain is both necessary and sufficient for the transformation response. Finally, using anti-Ese-1 monoclonal antibodies that we developed, we found Ese-1 to be expressed in the nucleus in established breast cancer cell lines, and we demonstrated that Ese-1 is required to maintain the transformed state by using shRNA knockdown of endogenous Ese-1 in these cells. Taken together, these studies reveal that Ese-1, as a sole agent, acts via a cytoplasmic mechanism to initiate transformation, but once mammary cells are fully transformed, via the action of several mechanisms, then nuclear Ese-1 is required to maintain the malignant phenotype.

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We are making a significant commitment to inducible-methods and transgenic approaches to decipher the role of Ras/MAPK signaling, and specific POU-homeodomain and Ets transcription factors in mediating the ontogeny, maintenance and tumorigenesis of epithelial cells in the pituitary and mammary systems.

Surface density representations (inlays: ribbon representation in the same orientation) of the crystal structure of the Pithd bound to DNA. These fragments were taken from the crystal structure as resolved by Jacobson et al (1997).

The results from figure 2C have been indicated by color coding. Chemical shift changes > 25 Hz are colored red, changes between 25 and 15 Hz dark red and between 15 and 10 pink. Unaffected residues are colored yellow. In green are the solvent exposed residues of the first helix, which showed a periodicity of three residues in chemical shift perturbation. The amide proton of W261, which is deeply buried, is colored in blue.

The hydrophobic pocket around W261 formed by V257, V258, I222 and W261 itself is colored in beige. Overlaying the pocket is K226, colored in blue. The DNA has been deleted for clarity.

Model for association of Ets-1 to the Pithd. K226 is shown in blue, the hydrophobic pocket in beige and T220 in green. Since the crystal structure does not extend beyond residue K273, an extended tail was added to represent the C-terminus of the Pithd. Ets-1 could use L288 for initial binding and subsequently, depending on the post-translational state of the Pithd, dock in the hydrophobic pocket next to W261. Additional stabilizing contacts may be made at the C-terminus of the DNA recognition helix (helix 3). These pictures were generated using MSI's Weblab viewer Lite version (www.msi.com).

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Bennett, W.F., Gutierrez-Hartmann, A. and Butow, R.A. The role of mitochondria-bound 80S ribosomes in mitochondrial biogenesis. IN: Interdisciplinary Conference on the Genetics and Biogenesis of Mitochondria and Chloroplasts. Amsterdam: North Holland Press, 1977.